Tumor necrosis factor (TNF) is a pleiotropic cytokine and a central mediator of inflammation. Elevated levels of TNF are associated with various inflammatory diseases including rheumatoid arthritis, psoriasis, and Crohn's disease. Several TNF-neutralizing reagents have been approved for the treatment of these diseases, including soluble TNF receptors (etanercept) as well as anti-TNF antibodies (infliximab, adalimumab, certolizumab pegol, golimumab), and many more are under development. With over 1 million patients treated with TNF antagonists, therapeutic efficacy is well documented. However, global TNF inhibition over a prolonged period of time increases the risk of tuberculosis reactivation, serious infections and even malignancies. Consequently, medical information of all approved anti-TNF medicines includes extensive warnings and precautions.
Two TNF receptors (CD120a, TNFR1 and CD120b, TNFR2) mediate signal transduction upon binding of TNF (Locksley et al., 2001, Cell 104:487-501). Pro-inflammatory responses are mainly mediated by the ubiquitously expressed TNFR1. TNFR1 is activated both by the membrane-bound form of TNF (mTNF) and soluble TNF (sTNF), which is produced from mTNF by proteolytic cleavage. In contrast, TNFR2, expressed in a more restricted manner e.g. by immune cells, endothelial cells and neurons, can only be activated by mTNF. Activation of TNFR2 mainly induces anti-apoptotic signals and can lead to cell proliferation in vitro. Furthermore, TNFR2 appears to play a role in tissue homeostasis and regeneration.
Selective inhibition of TNFR1 signaling has gained increasing attention as alternative to global TNF neutralization, which affects both TNF receptors (Fischer et al. 2015, Antibodies 4:48-70). Recently, a TNF mutein (R1antTNF) selectively neutralizing the activity of TNFR1 has been described (Shibata et al. 2008, Cytokine 2:229-33). This TNF mutein, administered either as unmodified or as PEGylated protein (PEG-R1antTNF), demonstrated therapeutic efficacy in acute murine hepatitis models and a murine collagen-induced arthritis model. The beneficial effect of selectively inhibiting TNFR1 was further supported by results from a dominant-negative TNF mutein (XPro1595), which is capable of forming inactive complexes with sTNF, thus selectively inhibiting the pro-inflammatory action mediated by TNFR1 while preserving the innate immunity to infections (Olleros et al. 2009, J. Infect. Dis. 199:1053-63).
TNFR1-selective inhibition can be also achieved with TNFR1-specific antibodies. For example, a monoclonal murine antibody, H398, and antibody described in U.S. Pat. No. 5,736,138, with selectivity for human TNFR1, showed potent inhibition of TNF-mediated signal transduction and cytotoxicity (Moosmayer et al. 1995, Ther. Immunol. 2:31-40).
A humanized version of H398 is described by WO2008/113515A2. Specifically a humanized antibody was produced as Fab fragment (IZI-06.1) and exhibited in vitro neutralizing activities comparable to that of the Fab fragment of the parental antibody. Importantly, the H398 antibody did not reach complete block of TNF activity, which was interpreted by the conversion from an antagonist into a partial agonist at high concentrations. This is explained by dose-dependent increase in TNFR1 crosslinking, thus potentially forming ligand-independent, functional TNFR1 signaling complexes.
Attempts towards affinity maturation of IZI-06.1 resulted in a mutant (scFvIG11) showing a two-fold increase in antigen binding affinity which also translated into slightly improved inhibition of TNF-mediated cytotoxicity in vitro (Zettlitz K A, thesis 2012, Universität Stuttgart).
Kontermann et al. (Journal Of Immunotherapy 2008, 31(3):225-234) describe a monovalent antibody fragment of IZI-06.1 as a TNFR1-selective TNF antagonist.
Antibodies to TNFR1 were found to have an agonistic potential by inducing a response mimicking the ligand. This response suggests that signal transduction is initiated by aggregation of receptors due to binding of the multivalent TNF trimers. In particular, divalent anti-TNFR1 antibodies were known to bear the risk of TNFR activation due to receptor crosslinking, causing themselves pro-inflammatory reactions, including cytotoxicity and apoptosis, which would be contraproductive in treating TNF-mediated disease conditions.
WO02012035141A1 describes an anti-huTNFR1 antibody of the IgG1 type called ATROSAB, which has a modified Fc region deficient in mediating effector function, which was found to limit the agonistic potential of the antibody.
Richter et al. (2013, PLoS One 8:e72156) describe the inhibition of TNFR1 to interact with its natural ligands TNF and lymphotoxin alpha (LTα) by ATROSAB as measured by the release of IL-6 and IL-8 from HeLa and HT1080 cells, respectively, induced by TNF or LTα.